截短型人骨保护素在CHO-DHFR^-细胞中的表达及活性测定  

Expression of truncated fragment of human OPG in CHO-DHFR^- cells and its bioactivity characterization

作　　者：阚伯红[1] 藏晓怡[1] 李兰英[1] 赵孔银[2] 

机构地区：[1]天津医科大学内分泌研究所卫生部及天津市激素与发育重点实验室,天津300070 [2]天津工业大学材化学院,天津300160

出　　处：《细胞与分子免疫学杂志》2009年第11期1002-1004,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：天津市自然科学基金重点项目(013803511)

摘　　要：目的:构建截短型人骨保护素(hTOPG)哺乳动物表达载体pcDNA3.1/DHFR-hTOPG,实现其在CHO-DHFR-细胞中的高效表达,获取生物活性较高的重组蛋白。方法:利用基因重组技术构建重组表达载体pcDNA3.1/DHFR-TOPG,按LipofectamineTM2000试剂盒说明书转染CHO-DHFR-细胞,以含50 mL/L透析血清的IMDM培养基培养,氨甲喋呤(MTX)加压筛选高表达细胞株,ELISA法和RT-PCR法测定重组蛋白和基因的表达,并采用破骨细胞样细胞(OLC)诱导分化抑制实验测定重组蛋白的体外活性。结果:重组蛋白的表达量最高可达6 mg/L.72 h,且能够明显抑制OLC生成(P<0.05)。结论:截短型人OPG在CHO-DHFR-细胞中成功高效表达,并具有良好的生物学活性,为进一步的实验研究和临床应用提供了基础。AIM. To obtain high level expression of recombinant human truncated osteoprotegerin （TOPG） with higher bioactivity in CHO-DHFR - cells. METHODS： The recombinant vector pcDNA3. I/DHFR-TOPG was constructed and transfected into CHO-DHFR- Cells by the directions of LipofectAMINETM 2000 for stable expression. The stable expression cell strains were screened by selective medium IM- DM with 50 mL/L FCS, then serially passed in methotraxate （MTX） for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro. RESULTS： The expression level of recombinant truncated human OPG was up to 6 mg/L ·72 h, and it had significant suppression effect on the formation of OLC（ P 〈 0.05）. CONCLUSION： Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.

关 键 词：骨保护素 CHO—DHFR-细胞 表达 活性分析 

分 类 号：R392.6[医药卫生—免疫学]